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RPA-IAC primer for detecting vibrio alginolyticus, kit, application and method thereof

A technology of vibrio alginolyticus and a kit is applied in the field of detecting vibrio alginolyticus in shellfish aquatic products, and achieves the effects of wide application range, high amplification efficiency and short reaction time

Inactive Publication Date: 2022-05-17
广州市微生物研究所集团股份有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] CN107227378A, CN107385057A, CN107227377A, CN106967816A, etc. have disclosed methods for detecting different species of Vibrio based on RPA-IAC. These methods have good accuracy, but they also further illustrate that RPA, especially RPA-IAC technology, still needs to be further improved

Method used

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  • RPA-IAC primer for detecting vibrio alginolyticus, kit, application and method thereof
  • RPA-IAC primer for detecting vibrio alginolyticus, kit, application and method thereof
  • RPA-IAC primer for detecting vibrio alginolyticus, kit, application and method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0053] This embodiment provides the RPA primer pair and the amplification internal standard sequence and its application.

[0054] The screening method for the RPA primer pair is as follows: Aiming at the Vibrio alginolyticus collagenase gene (GenBank ID: CP012739.1), multiple RPA primers were designed and screened in large quantities, and their specificity, sensitivity, and the interaction between primers were integrated. , and the adaptability of the primers used to the RPA amplification kit, and finally screened out the following RPA primer pairs with good specificity, good repeatability and high sensitivity that can detect Vibrio alginolyticus:

[0055] collagenase collagenase-F: 5'-GCTTATCGAGTCGATGCGCCTGACAAGTGATC-3' (SEQ ID NO: 1);

[0056] collagenase collagenase-R: 5'-TCATAAGGGTCCCGTATGCGTACTTGCTAT-3' (SEQ ID NO: 2);

[0057] Design and synthesis of amplified internal standard sequence: In order to avoid homologous interference of similar bacteria and individual nucle...

Embodiment 2

[0062] This embodiment provides a kit and application thereof, the kit comprising:

[0063] (1) SEQ ID NO: 1, SEQ ID NO: 2 (both from Example 1), and a plasmid containing the amplified internal standard sequence shown in SEQ ID NO: 3;

[0064] (2) DNA extraction reagents;

[0065] (3) tube containing freeze-dried enzyme powder;

[0066] (4) rehydration buffer;

[0067] (5) Magnesium acetate solution.

[0068] The above tubes containing lyophilized enzyme powder, rehydration buffer (Rehydration Buffer) and magnesium acetate solution (280mmol / L) are reagents from the RPA amplification kit TwistAmp Basic kits (TwistDX company, product number TABAS03KIT).

[0069] The DNA extraction reagent is the reagent included in the animal genome DNA extraction kit from Tiangen Biochemical Technology Co., Ltd. whose article number is DP432 and the bacterial genome DNA extraction kit from Tiangen Biochemical Technology Co., Ltd. whose article number is DP302. .

[0070] Construction of th...

Embodiment 3

[0076] This embodiment provides a method for detecting or assisting the detection of Vibrio alginolyticus, such as detecting or assisting in detecting whether a biological sample is infected with Vibrio alginolyticus, or detecting or assisting in detecting whether the pathogen is or is a candidate for Vibrio alginolyticus, the method The primer pair of Example 1 or the kit of Example 2 were used.

[0077] Above-mentioned method comprises the steps:

[0078] Step 1: RPA-IAC amplification

[0079] Using the DNA of biological samples or pathogenic bacteria as a template, add the plasmid containing the amplified internal standard sequence shown in SEQ ID NO: 3 in Example 2, and use the primer pair consisting of SEQ ID NO: 1 and SEQ ID NO: 2 to perform RPA -IAC amplification to obtain the RPA-IAC amplification product, while setting a blank control (the template is ultrapure water).

[0080] The preparation method of the RPA-IAC amplification system is as follows: Add 29.5 μL of ...

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Abstract

The invention discloses an RPA-IAC primer for detecting vibrio alginolyticus, a kit, application and a method thereof. The RPA primer pair is shown as SEQ ID NO.1 and SEQ ID NO.2. The invention also discloses a method for detecting the vibrio alginolyticus. The RPA primer pair disclosed by the invention has good amplification efficiency and amplification specificity on vibrio alginolyticus, and the sensitivity of RPA detection can be improved; the method is simple in operation, does not need special thermal cycle equipment, is short in reaction time, and compared with LAMP, an amplification product does not have a dispersion band, an RPA-IAC amplification product has a band with a specific size according to a primer design site, and a result is easy to judge. And by adding an amplification internal standard, a false negative result is effectively avoided.

Description

technical field [0001] The invention relates to the field of biological detection, in particular to a method, a primer and a kit for detecting vibrio alginolyticus in shellfish aquatic products. Background technique [0002] Vibrio alginolyticus is a non-encapsulated Gram-negative halophilic Vibrio, which is widely distributed in shellfish and crustaceans such as oysters, shrimps, and crabs cultured in seawater, and is the most prevalent and harmful food One of the source pathogens. [0003] At present, there are reports of Vibrio alginolyticus in most coastal countries in the world. There are also more reports of infection in Jiangsu, Zhejiang, Fujian and Guangdong coastal areas, and there are several cases of Vibrio alginolyticus infection, which is an important issue for public health. One of the threats, so the detection of Vibrio alginolyticus is of great significance in public health. [0004] At present, the detection of Vibrio alginolyticus still mainly relies on t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6844C12N15/11C12R1/63
CPCC12Q1/689C12Q1/6844C12Q2600/166C12Q2521/507C12Q2522/101C12Q2531/119C12Q2549/10Y02A50/30
Inventor 蒋丽婷杨础华赵培静孙崇臻李小凤黄东浪刘蔓蔓林秋霞甘祥武文晓欣
Owner 广州市微生物研究所集团股份有限公司
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